The interaction of multivalent cations, particularly calcium, with biological membranes is of primary importance in many biological phenomena, such as nerve excitability, regulation of muscle contraction, exocytotic discharge of chemical transmitters, blood clotting, and mineralization of tissue. In this project the interactions of such ions with the phospholipid component of biological membranes will be studied, both experimentally and theoretically. Emphasis will be placed on quantitative elucidation of the binding of calcium to membranes of phosphatidylinositol (PI) and phosphatidylserine (PS), which are the major negatively-charged phospholipids of intracellular membranes and of cell plasma membranes, respectively. PI is also involved in the mobilization of intracellular Ca++. The aim is to provide answers to the following fundamental questions: (1) What is the ion:phospholipid stoichiometry of Ca++ binding to PI and PS? (2) Do anions bind to PI and PS membranes? (3) Does Ca++ compete with monovalent cations for PI and PS binding sites? (4) Does Ca++ compete with or displace protons from PI and PS binding sites? The same questions will be addressed to membranes made of PI and PS mixtures with neutral phospholipids, to membranes containing phosphorylated PI derivatives, to lipids extracted from sacroplasmic reticulum membranes, and to divalent cations other than Ca++. Experiments will be of three types: (1) Electrostatic surface potentials of planar bilayer lipid membranes will be monitored by use of monazomycin, a surface-potential-sensitive conductance probe. (2) Particle electrophoresis will be used to determine conditions under which phospholipid vesicles are exactly neutralized by adsorbed ions. (3) pH-stat and Ca-stat measurements will detect Ca++-H+ competition effects. The parameters to be varied in all experiments are: (a) multivalent-cation concentration and species, (b) monovalent-cation concentration and species, (c) anion concentration and species, including chaotropic anions, (d) pH, (e) membrane charge density, (f) lipid species. Conditions designed to optimize experimental sensitivity to each of the questions posed above will be employed. Data will be analyzed by use of a theory, previously published by the applicant, that treats both 1:1 and 1:2 divalent-cation binding stoichiometries as well as both competitive and non-competitive binding of divalent and monovalent cations to phospholipid membranes. The theory will be extended to include anion and proton binding phenomena.